Stereoselective Synthesis of β-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution**

Article abstract of DOI:10.1002/anie.202105656

β-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic β-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp³-rich cyclic fragments and the first total synthesis of jomthonic acid A.

Full text of DOI:10.1002/anie.202105656

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Stereoselective Synthesis of b-Branched Aromatic a-Amino Acids
via Biocatalytic Dynamic Kinetic Resolution
Authors: Fuzhuo Li, Li-Cheng Yang, Jingyang Zhang, Jason Chen, and
Hans Renata
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202105656
Link to VoR: https://doi.org/10.1002/anie.202105656

10.1002/anie.202105656
Angewandte Chemie International Edition
COMMUNICATION
b
a
Stereoselective Synthesis of -Branched Aromatic -Amino Acids
via Biocatalytic Dynamic Kinetic Resolution
Fuzhuo Li,[a] Li-Cheng Yang,[a] Jingyang Zhang,[a] Jason S. Chen,[b] Hans Renata*[a]
Abstract: b-branched noncanonical amino acids are valuable
A
O
Me
O
molecules in modern drug development efforts. However, they are still
challenging to prepare due to the need to set multiple stereocenters
in a stereoselective fashion and contemporary methods to achieve
this often relies on the use of rare transition metal catalysis with
designer ligand. Here, we report a biocatalytic transamination method
to prepare a broad range of aromatic b-branched a-amino acids that
proceeds with high diastereo- and enantioselectivity. Mechanistic
studies show that the transformation proceeds through dynamic
kinetic resolution that is unique to the optimal enzyme. To highlight its
utility and practicality, the biocatalytic reaction is applied to the
synthesis of several sp3-rich cyclic fragments and in the first total
synthesis of jomthonic acid A.
Me
NH
Me Me
O
tBu
O
NH
Me
NH
N
CO2Me
Me
N
O
N
H
HN
H
N
F
SO2
N
N
S
tBu
N
H
O
H2NOC
O
R1
CONMe2
Cl
bottromycin A2 (1a): R1 = Me;
MIC (MRSA70) = 1 µg/mL
2: RNR inhibitor, IC50 = 40 nM
1b: R1 = H; MIC (MRSA70) > 32 µg/mL
HO
3a: R2 = Me;
R2
Ki = 0.97 nM, Kiδ = 6.4 µM
3b: R2 = H;
µ
N
H
N
H2N
Ki = 4.0 nM, Kiδ = 2.7 µM
µ
O
CONH2
N
H
O
O
β-MePhe analogue modulates µ opioid
selectivity via conformational constraint
Amino acids represent one of the most indispensable and
versatile building blocks in modern drug discovery.1 In addition to
its a-amino and carboxylate groups, each amino acid also
contains a signature side chain that provides unique three-
dimensionality and an additional structural motif for modular
diversification of peptides in combinatorial synthesis. To
complement the 20 canonical amino acids used in protein
biosynthesis, nature also employs a variety of tailoring reactions
such as hydroxylation, halogenation and methylation to further
diversify these building blocks.2 Such modification serves to
modulate the physicochemical properties of the resulting
noncanonical amino acid,3 as well as the final oligopeptide which
incorporates such motif (Figure 1A). Of particular note are
noncanonical amino acids (ncAAs) that contain an additional
stereogenic center at the b-position due to the synergistic effects
of the two adjacent stereocenters to confer additional structural
rigidity. For example, the presence of a b-methylphenylalanine (b-
MePhe) motif in bottromycin A2 (1a) was found to be vital in
conferring inhibitory activity towards the prokaryotic 30S
ribosomal subunit as the desmethyl analogue of the natural
product (1b) was found to be a poor antibiotic.3a An analogue of
endomorphin bearing additional methylation at the b position of its
Phe units (3a) was also shown to exhibit significantly improved
potency and selectivity for the d opioid receptor relative to the µ
opioid receptor.3b
B. Chemical synthesis of β-branched amino acids
asymmetric hydrogenation
C–H activation approach
Me
NHPhth
CO2Me
Me
CONHArF
NHAc
Me
Pd(TFA)2
ligand
Ag2CO3, Δ
H2, Rh cat.
chiral phosphine
N
O
Me
ligand
Me
NHPhth
CO2Me
Me
CONHArF
NHAc
Ph
C. This work – biocatalytic dynamic kinetic resolution
amino acid
aminotransferase
R
O
R
O
OH
OH
Ar
Ar
high diastereoselectivity
high enantioselectivity
O
NH2
enantioenriched product
achiral substrate
Figure 1. A. Select examples of bioactive molecules containing b-branched
aromatic amino acids. B. Contemporary strategies to prepare b-branched
aromatic amino acids via transition metal catalysis. C. Proposed biocatalytic
synthesis of b-branched aromatic amino acids via diastereoselective
transamination of a-ketoacids.
Despite their utility in modern peptide drug discovery, b-branched
ncAAs remain highly challenging to synthesize due to the need
to construct multiple stereocenters. Chemical synthesis of ncAAs
emphasizes on the use of asymmetric transformations to set the
a-stereocenter (Figure 1B) and has resulted in the development
of several practical strategies, including asymmetric
hydrogenation,4 asymmetric Strecker reaction,5 and the use of
chiral auxiliaries in either polar-6 or radical-based7 reactions.
However, synthesis of b-branched ncAAs using any of these
[a]
Fuzhuo Li,[+] Li-Cheng Yang,[+] Jingyang Zhang, Prof. Dr. Hans
Renata
Department of Chemistry
The Scripps Research Institute
130 Scripps Way, Jupiter, FL 33458
E-mail: hrenata@scripps.edu
These authors contributed equally to this work.
Jason S. Chen
[+]
[b]
Automated Synthesis Facility
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037
strategies will require
a separate step to establish the
stereocenter at the b position. In the case of radical-based
method, facile racemization at the radical center leads to a 1:1
diastereomeric mixture at the b position. Recent advances in C–
H functionalization have enabled the synthesis of b-branched
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202105656
Angewandte Chemie International Edition
COMMUNICATION
ncAAs through direct arylation or alkylation of the b-carbon.8
However, such approaches often require the use of rare transition
metal catalysts at high catalyst loading and pre-installation of a
directing group and at times, suffer from sub-optimal
diastereoselectivity.
on hormaomycin.17 Importantly, these observations led us to
hypothesize that ArATs are capable of accepting related pyruvate
substrates that contain additional substituents at the b position to
produce b-branched aromatic amino acids and that we would be
able to identify a suitable ArAT that could catalyze this process
with high diastereoselectivity.
Enzymatic transformations are becoming widely applied in both
academia and industry.9 By virtue of their unparalleled selectivity
profile, they represent an attractive solution to the challenges
associated with ncAA synthesis.10 Indeed, a gamut of biocatalytic
processes has been developed in recent years in this area.
Nevertheless, only a handful of these methods allow for the
generation of multiple stereocenters with high enantio- and
diastereoselectivity in a single transformation. Arnold and co-
workers recently reported the use of engineered tryptophan
synthases for the formation of b-alkyl tryptophan analogues.11
However, this approach is limited to indole-containing b-branched
ncAAs. Similarly, Poelarends and co-workers have engineered
methylaspartate ammonia lyases for the production of branched
aspartate derivatives.12 Seebeck and co-workers13 also described
the use of a self-contained enzymatic cascade for asymmetric b-
methylation of a-amino acids, though this method requires the
use of three enzymes in the cascade at high enzyme loading (2
mol% loading) and proceeds with low total turnover numbers
overall. Gefflaut and co-workers have also reported the use of
aminotransferases in the preparation of branched glutamate
analogues,14 but these reactions proceeded either via traditional
kinetic resolution or with poor diastereoselectivity.
Here, we report a biocatalytic dynamic kinetic resolution (DKR)
approach for the synthesis of b-branched aromatic amino acids
that (1) establishes two contiguous stereocenters with complete
diastereocontrol, (2) proceeds with excellent enantioselectivity
towards the L-amino acid product and high catalyst efficiency, and
(3) employs readily available a-ketoacid substrates (Figure 1C).
Key in the reaction design is the identification of a suitable
thermophilic enzyme that is able to withstand the non-
physiological conditions required while also exhibiting several
unique features to enable the realization of a DKR process. To
the best of our knowledge, this is the first report of a biocatalytic
DKR process for the production of noncanonical a-amino acids.
While w-transaminases15 have previously been used in DKR
processes including in the preparation of active pharmaceutical
ingredients, applications that proceed with high diastereo- and
enantioselectivity are still few and far between.
Due to the importance of aromatic amino acids, ArAT is present
in all domains of life and TyrB homologs have been identified from
various species. Nevertheless, these ArATs have enjoyed only
limited biocatalytic application. We began our investigation by
examining the synthetic utility of a panel of TyrB homologs in the
conversion of phenylpyruvate to b-MePhe. Of special interest are
ArATs belonging to thermophilic bacteria due to the well-known
benefits associated with thermostable enzymes in biocatalysis,
namely the ability to withstand harsh reaction conditions, as well
as superior evolvability18 for future engineering efforts. With this
feature in mind, three thermophilic enzymes, TlArAT (from T.
litoralis),19 PhArAT (from P. horikoshii)20 and TtArAT (from T.
thermophilus),21 were included in our initial screening. In addition
to their thermophilicity, these three enzymes have also been
structurally characterized, though their use in biotechnology has
not been explored before.
Our initial screening with b-methylphenylpyruvic acid (4a, Figure
2A) revealed that while transamination with TyrB was able to
deliver the desired product in 60% yield (total turnover number,
TTN = 750), it proceeded with poor diastereoselectivity (dr =
1.5:1). A similar observation was obtained in reactions with
PdArAT (from P. denitrificans)22 and TlArAT, whereby product 5a
was obtained only in 32% and 35% yields and moderate to poor
diastereoselectivity. In contrast, reactions with PhArAT and
TtArAT provided excellent diastereoselectivity for the desired
product, though only moderate yields were observed. Further
optimization with PhArAT improved the yield of 5a to 56% at 60
ºC but this improvement was accompanied by a slight decrease
in diastereoselectivity. In contrast, TtArAT delivered 5a in 74%
yield at 40 ºC and pH 9.0 without any loss in diastereoselectivity.
Preliminary investigations also showed that TtArAT is more
promiscuous than PhArAT and the former was chosen for
subsequent investigation. As a benchmark, we also tested
phenylalanine ammonia lyase (PAL) from A. variabilis23 and
phenylalanine dehydrogenases (PheDHs)24 from B. sphaericus
and C. thermarum for the preparation of 5a, but all reactions failed
to provide the desired product.
Aromatic amino acid aminotransferases (ArATs) are pyridoxal-
phosphate (PLP)-dependent enzymes that are responsible for the
biosynthesis
of
phenylalanine
via
transamination
of
phenylpyruvate with other amino acids as the amine donor.
Several lines of evidence from the biosynthetic literature hint at
the promiscuity of these aminotransferases. In their investigation
on the biosynthetic origins of the b-MePhe moiety of
mannopeptimycin,16 Li and co-workers were able to identify a
dedicated methyltransferase that methylates phenylpyruvate at
the b position but were not able to find an aminotransferase within
the biosynthetic gene cluster. Hypothesizing that an enzyme from
primary metabolism is responsible for the latter transformation,
the authors showed that TyrB, an ArAT from E. coli, is capable of
converting b-methylphenylpyruvic acid to b-MePhe, albeit as a
diastereomeric mixture at the b position. A similar observation
was also made by Piel and co-workers in their biosynthetic studies
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10.1002/anie.202105656
Angewandte Chemie International Edition
COMMUNICATION
different types of kinetic resolution process that are operative with TyrB and
TtArAT through mechanistic studies: transamination of 4a with TtArAT proceeds
through dynamic kinetic resolution featuring facile enantiomer interconversion,
followed by selective transamination of one of the enantiomers.
A
ArAT
(0.08 mol%),
Glu or Gln
Me
O
Me
5a
O
OH
OH
PLP,
pH 8, rt
O
NH2
pH 9.0,
40 ºC
4a
74%
dr > 20:1
80
60
40
20
0
That conversion of more than 70% could be observed with 4a
suggested that a DKR process might be operative with TtArAT. In
this mechanism, the two substrate enantiomers could be rapidly
interconverting but only one could preferentially undergo
productive reaction with TtArAT. Prior studies in synthetic
methodology, biocatalysis and mechanistic enzymology have
also demonstrated that stereocenters adjacent to carbonyl groups
are relatively labile under ambient conditions.15,25 To verify this
hypothesis, we conducted further mechanistic studies with
TtArAT while using TyrB as a benchmark to uncover any potential
mechanistic dichotomy. First, pure 3S and 3R enantiomers of 4a
(4a-Ent1 and 4a-Ent2 respectively) were obtained via preparative
chiral SFC separation and submitted to reactions with TyrB and
TtArAT (Figure 2B). Transamination of 4a-Ent1 with TyrB led to
only minor formation of the syn diastereomer 5a, with the anti
diastereomer formed as the major product. Additionally, the
reaction became less diastereoselective at elevated temperature
or pH. In contrast, TtArAT-catalyzed transamination of 4a-Ent1
proceeded with stereoinversion at C3, providing the syn
diastereomer 5a exclusively. As expected, all reactions with 4a-
Ent2 formed syn 5a as the major diastereomer regardless of the
enzyme used. Using deuterium incorporation rate at the b-position
of racemic 4a in the absence of enzyme as a proxy for
racemization rate, we noted that deuterium incorporation could
take place at pH 8 and became more prominent at increased pH
and temperature (See Supporting Information Figure S1). This
observation suggested that the two substrate enantiomers are
able to interconvert–albeit at different rates–under all reaction
conditions tested with TyrB and TtArAT. However, TyrB shows
only limited ability to discriminate between the enantiomers in its
active site. While racemization of substrate enantiomers takes
place more rapidly under optimal conditions with TtArAT,
productive catalysis with the enzyme only takes place with the 3R
enantiomer. Further mechanistic and structural studies to
elucidate the finer details of this process are ongoing.
60%
dr = 1.5:1
60 ºC
56%
dr = 10:1
46%
dr > 20:1
44%
dr > 20:1
35%
dr = 1:1
32%
dr = 4.3:1
TyrB PdArAT TlArAT PhArAT TtArAT PhArAT TtArAT
ArAT variant
Comparison with other biocatalytic methods
PAL,
NH3
Me
O
Me
O
OH
OH
NH2
PheDH,
NH3
NADH
Me
O
Me
O
OH
OH
O
O
NH2
B. Mechanistic studies
Me
ArAT
(0.08 mol%),
Glu or Gln
Me
5a
O
OH
OH
PLP,
pH, T
O
NH2
4a-Ent1 (3S, 100% ee)
Enzyme (pH, T)
5a yield (%)
5a dr
TyrB (8.0, rt)
TyrB (8.0, 40 ºC)
TyrB (9.0, 40 ºC)
TtArAT (9.0, rt)
33%
45%
15%
33%
21%
1:7–1:10
1:5.5
1:5
Following optimization, the scope and limitations of the
transformation were tested on various substrates (Figure 3A).
Productive reactions were observed with a variety of substrates
bearing additional functional groups on their aromatic ring. In
general, substitution at the para position on the ring (relative to
the amino acid alkyl chain) is more tolerated than that at the ortho
and meta positions. In several cases, the use of elevated reaction
temperature (60 °C) was found advantageous in improving the
>20:1
>20:1
TtArAT (9.0, 40 ºC)
R
O
R
O
slow
OH
OH
Ar
Ar
NH2
O
reaction yields, demonstrating the benefit of employing
a
R
O
R
O
fast
thermophilic enzyme in the reaction. Comparison of reaction
yields obtained for products 5k–m, 5o, and 5q suggests slight
preference for substrates bearing electron withdrawing groups.
However, no strong correlation between the Hammett parameters
of the respective ring substituents and yields could be observed.
Thus, any variation in activity likely arises primarily due to
differences in steric interactions within the active site. Interestingly,
increasing the size of the aryl ring to a naphthyl group led to only
a small decrease in reaction yield. In cases with lower yields, non-
OH
OH
Ar
Ar
NH2
O
dynamic kinetic resolution is uniquely
operative in reaction with TtArAT
Figure 2. A. Screening of various ArATs for the transamination of 4a and
comparison with other biocatalytic methods. TyrB: L-ArAT from E. coli, PdArAT:
L-ArAT from P. denitrificans, TlArAT: L-ArAT from T. litoralis, PhArAT: L-ArAT
from P. horikoshii, TtArAT: L-ArAT from T. thermophilus, PAL: phenylalanine
ammonia lyase, PheDH: phenylalanine dehydrogenase. B. Verification of the
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10.1002/anie.202105656
Angewandte Chemie International Edition
COMMUNICATION
enzymatic decarboxylation of the substrate was observed to be
the main competing pathway.
No reaction
A
5a: 74%, >20:1 dr, 99% ee
H
TtArAT,
PLP,
Gln
O
R
O
R
O
5b: 70% (60 ºC), >20:1 dr, 94% ee
5c: 77% (60 ºC), >20:1 dr, 94% ee
5d: 63%, >20:1 dr, 96% ee
F
OH
OH
OH
O
Ar
Ar
Cl
Me
40 ºC,
pH 9.0
4x
O
NH2
Me
O
Me
O
OH
Me
5e: 73%, >20:1 dr, 95% ee
F
OH
R1
Me
O
O
4y
NH2
5f: 75%, >20:1 dr, 93% ee
Cl
R2
R3
O2N
OH
Me
Me
5s: 98%, >20:1 dr, 98% ee
O
O
5g: 64% (60 ºC), >20:1 dr, 93% ee
5h: 58% (60 ºC), 10:1 dr, 93% ee
CF3
NO2
NH2
Me
O
Me
O
OH
OH
F
F
F
OH
O
4z
OH
NH2
5i: 83%, >20:1 dr, 95% ee
5j: 70%, >20:1 dr, 92% ee
5k: 89%, >20:1 dr, 94% ee
5l: 95%, >20:1 dr, 99% ee
5m: 97%, >20:1 dr, 93% ee
5n: 90%, >20:1 dr, 92% ee
5o: 95%, >20:1 dr, 94% ee
5p: 86%, >20:1 dr, 95% ee
5q: 67%, >20:1 dr, 95% ee
5r: 70%, >20:1 dr, 93% ee
F
I
NH2
Me
5u: 95% (60 ºC),
>20:1 dr, 93% ee
F
5t: 95%, >20:1 dr, 94% ee
Cl
CF3
Me
OMe
Me
Me
O
O
O
Br
NO2
Ac
4aa
OH
OH
Me
O
NH2
NH2
OH
5v: 86%, >20:1 dr,
5w: 56%, >20:1 dr,
4ab
O
93% ee
93% ee
B. Synthesis of sp3-rich fragments
Me Me
C. Cross-coupling of 5n
D. Natural product total synthesis
Gln, PLP,
TtArAT (lysate)
pH 9 Tris, 40 ºC
Me
O
Me
O
Me
O
NPhth
NPhth
PIFA, 0 ºC
OH
OH
Me
OH
56%, 500 mg scale
66%, 200 mg scale
>20: 1 dr, 99% ee
64%
O
NHR
HN
O
N
O
NH2
Boc2O,
NaHCO3
OMe
OMe
5a; R = H
14; R = Boc
Me
7
8
4a
11
LDA, MeI
Me
3 steps
90%
o-tolB(OH)2,
[PdCl2(dppf)]
CO2Me
46%
CO2Me
HO
Me
Me
1. Ph(CO)2O
2. (COCl)2
HO
Me
O
76%
DCC,
DMAP
87% over
2 steps
methyl (R)-3-
hydroxybutyrate
Me
Me
O
single diastereomer
15
NPhth
OH
(18)
3. AlCl3
OH
NH2
5a
Me
Me
O
Me
O
6
NH2
60% over 3 steps
CO2H
methylation,
DG
installation
I
CO2Me
5n
78%
O
vinylBF3K,
[PdCl2(dppf)]
HOBt, DMAP, EDCI
89% over 2 steps
86%
NHR
Me
Me
N
Me
O
16; R = Boc
17; R = H
HCl,
dioxane
Pd(OAc)2,
PIDA
OMe
CO2Me
Me
O
Me
Me
O
H
HN
O
Me3SnOH,
DCE, 80 ºC, 70%
Me
N
CO2R
O
O
56%
OH
N
19; R = Me
Me
O
N
9
NH2
Me
jomthonic acid A (13);
10
12
R = H
• first synthesis of 13
Figure 3. A. Substrate scope of biocatalytic transamination with TtArAT. Reaction conditions: a-ketoacid (10 mM, 1 equiv), Gln (30 mM, 3 equiv), PLP (0.25 mM,
2.5 mol%), TtArAT (0.008 mM, 0.08 mol%), Tris buffer (pH 9.0, 50 mM, 15 mL total volume), 24 h at 40 or 60 °C. Yields refer to isolated yields after C18 purification.
See Supporting Information for details on dr and ee measurements. B. Derivatization of b-MePhe (5a) for the synthesis of several sp3-rich fragments. C.
Diversification of product 5n through Pd-catalyzed Suzuki coupling. D. Application of biocatalytic transamination with TtArAT in the total synthesis of jomthonic acid
A (13).
position of the substrate. At present, the transamination reaction
The enzymatic transformation is also well-suited for the
production of b-MePhe analogues with multiple substituents (e.g.
5s–u) in high yields. This feature is expected to be useful for
further derivatization and manipulation of the aryl ring to arrive at
more complex structures. While a small change from methyl to
ethyl at the b-position is tolerated, more drastic deviations such
as the introduction of a propyl, cyclopropyl and isopropyl at this
position led to no reaction. This observation suggests that the
active site of TtArAT is highly sensitive to steric effects at the b
is limited to the production of b-branched Phe analogues as
substrates containing aliphatic chains or heteroaromatics did not
participate
in
the
reaction.
Nevertheless,
excellent
diastereoselectivity and enantioselectivity were observed in all
productive reactions. Another attractive feature of this method is
the ability to attain high conversion and yield with a single enzyme
system. Here, the need for L-glutamine as the amine donor with
TtArAT is particularly enabling as the resulting 2-oxoglutaramate
byproduct is known to undergo intramolecular cyclization and
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10.1002/anie.202105656
Angewandte Chemie International Edition
COMMUNICATION
drive the transamination equilibrium forward.26 In contrast, other
approaches to prepare ncAA through biocatalytic reductive
amination or transamination often require the use of additional
enzymes for cofactor recycling, amine donor recycling or
byproduct removal to drive the reaction equilibrium forward.17
We next sought to showcase the synthetic utility and versatility of
this biocatalytic platform in the production of various sp3-rich
cyclic fragments (Figure 3B). Such complex structures are rich in
three-dimensionality and are becoming increasingly valuable
building blocks to “escape from flatland” in combinatorial
synthesis and drug discovery.27 Following appropriate protecting
group introduction, a derivative of 5a readily underwent a Friedel-
Crafts cyclization28 to generate an indanone product containing
two stereocenters (6). Introduction of N-methoxyamide auxiliary
on 5a facilitated an oxidative cyclization in the presence of
[bis(trifluoroacetoxy)iodo]benzene (PIFA)29 to afford a multiply
substituted 3,4-dihydroisoquinoline product (8). In a similar
fashion, a chiral indoline containing two defined stereocenters
(10) could be synthesized through the use of palladium-catalyzed
C–H amination approach developed by Chen.30 The ability to
produce halogen-containing b-MePhe derivatives using this
method also facilitated the synthesis of more complex products
through metal-catalyzed cross-coupling (Figure 3C).31 For
example, the use of Suzuki coupling on unprotected 5n readily
afforded biaryl product 11 or styrenyl product 12.
other types of aminotransferases should also enable access to
alternative product stereoisomers. Further studies in these areas
towards the biocatalytic synthesis of more complex branched
amino acids are actively being pursued in our laboratory.
Experimental Section
See Supporting Information for Experimental Details.
Acknowledgements
Financial support for this work is generously provided by The
Scripps Research Institute and the National Institutes of Health
(grant R35 GM128895). We thank K. M. Engle for helpful
discussions on our mechanistic studies. We acknowledge B. B.
Sanchez, E. J. Sturgell, A. Romine and L. Oxtoby for technical
assistance in SFC separation and analysis. We thank the Shen
lab and the Bannister lab for generous access to their
instrumentations.
Keywords: noncanonical amino acid • transaminase •
biocatalysis • dynamic kinetic resolution
Finally, we demonstrated the viability and practicality of this
method for preparative-scale production of b-MePhe (5a) to meet
the material supply demands of a total synthesis campaign
(Figure 3D). Here, jomthonic acid A (13), a soil-derived natural
product with antidiabetic and antiatherogenic activities,32 was
chosen as synthetic target. Our approach commenced with the
use of TtArAT-catalyzed transamination to produce 5a on more
than 500 mg scale in 56–66% yield. For subsequent synthetic
manipulations, 5a was submitted to a routine Boc protection. In
parallel, alcohol 15 was prepared via a diastereoselective a-
methylation of methyl (R)-3-hydroxybutyrate.33 Coupling of 14 and
15 in the presence of DCC and DMAP proceeded uneventfully to
afford ester 16, which was treated with HCl in dioxane to unmask
its free amine. Following peptide coupling of 17 with acid 18,
selective methyl ester hydrolysis was achieved through the use of
Me3SnOH to complete the first synthesis of jomthonic acid A.
In conclusion, by leveraging the intrinsic sequence diversity of
ArATs, we identified a suitable thermophilic ArAT for the
biocatalytic production of b-branched aromatic amino acids,
which establishes two adjacent stereocenters with high
stereoselectivity in a single transformation through a unique DKR
process. The transformation is highly efficient and practical,
enabling further diversification of the products obtained to
generate sp3-rich fragments for potential applications in drug
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Entry for the Table of Contents
COMMUNICATION
Fuzhuo Li, Li-Cheng Yang, Jingyang
Zhang, Jason S. Chen, Hans Renata*
amino acid
aminotransferase
R
O
R
O
OH
OH
Ar
Ar
Page No. – Page No.
O
NH2
Stereoselective Synthesis of
b
-
enantioenriched product
racemic substrate
Branched Aromatic -Amino Acids
a
via Biocatalytic Dynamic Kinetic
Resolution
Me
O
Me
H
N
Me
O
Me
CO2H
O
Me
OH
O
Me
Me
jomthonic acid A
• first total synthesis
NH2
O2N
98%, >20:1 dr, 98% ee
Dynamic Transamination: A transaminase-based dynamic kinetic resolution was developed for the preparation of b-branched
aromatic a-amino acids with high diastereo- and enantioselectivity. The reaction was facilitated by the use of a thermophilic enzyme
that tolerates elevated temperatures and pH and exhibits broad substrate promiscuity. The utility of the process was demonstrated in
the first synthesis of jomthonic acid A.
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